U.S. patent application number 16/770906 was filed with the patent office on 2021-06-10 for bzip transcription factors regulate conversion of nicotine to nornicotine.
The applicant listed for this patent is R.J. REYNOLDS TOBACCO COMPANY, University of Kentucky Research Foundation. Invention is credited to Darlene Madeline Lawson, Sitakanta Pattanaik, Sanjay K. Singh, Ling Yuan.
Application Number | 20210171967 16/770906 |
Document ID | / |
Family ID | 1000005431537 |
Filed Date | 2021-06-10 |
United States Patent
Application |
20210171967 |
Kind Code |
A1 |
Yuan; Ling ; et al. |
June 10, 2021 |
bZIP TRANSCRIPTION FACTORS REGULATE CONVERSION OF NICOTINE TO
NORNICOTINE
Abstract
A method of decreasing conversion of nicotine to nornicotine is
provided herein. The methods includes administering at least one
basic region/leucine zipper (bZIP) type transcription factor
inhibitor to an organism in need thereof. Also provided herein is a
method of decreasing conversion of nicotine to nornicotine
including mutating a bZIP type transcription factor binding site on
a promoter of a nicotine N-demethylase (NND). Further provided
herein is a method of decreasing conversion of nicotine to
nornicotine including mutating a plant genome to knockout at least
one bZIP type transcription factor.
Inventors: |
Yuan; Ling; (Lexington,
KY) ; Singh; Sanjay K.; (Lexington, KY) ;
Pattanaik; Sitakanta; (Lexington, KY) ; Lawson;
Darlene Madeline; (Kernersville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Kentucky Research Foundation
R.J. REYNOLDS TOBACCO COMPANY |
Lexington
Winston-Salem |
KY
NC |
US
US |
|
|
Family ID: |
1000005431537 |
Appl. No.: |
16/770906 |
Filed: |
December 6, 2018 |
PCT Filed: |
December 6, 2018 |
PCT NO: |
PCT/US2018/064317 |
371 Date: |
June 8, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62595983 |
Dec 7, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/11 20130101;
C12N 2310/3231 20130101; C12N 15/113 20130101; C12N 15/8218
20130101; C12N 2310/141 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/113 20060101 C12N015/113 |
Claims
1. A method of decreasing conversion of nicotine to nornicotine,
the method comprising administering at least one basic
region/leucine zipper (bZIP) type transcription factor inhibitor to
an organism in need thereof.
2. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor is selected from the group
consisting of group C bZIP transcription factor inhibitors, group S
bZIP transcription factor inhibitors, and combinations thereof.
3. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor is selected from the group
consisting of an NtbZIP1a inhibitor, an NtbZIP1b inhibitor, an
NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations
thereof.
4. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor comprises an NtbZIP1a inhibitor and
an NtbZIP1b inhibitor.
5. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor comprises an NtbZIP2a inhibitor and
an NtbZIP2b inhibitor.
6. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor comprises an NtbZIP1a inhibitor, an
NtbZIP1b inhibitor, an NtbZIP2a inhibitor, and an NtbZIP2b
inhibitor.
7. The method of claim 1, wherein the at least one bZIP
transcription factor inhibitor is selected from the group
consisting of antisense oligonucleotides, miRNA, siRNA, locked
nucleic acid (LNA) nucleotides, and combination thereof.
8. The method of claim 7, wherein the at least one bZIP
transcription factor inhibitor comprises an antisense
oligonucleotide of a bZIP transcription factor selected from the
group consisting of NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and
combinations thereof.
9. A method of decreasing conversion of nicotine to nornicotine,
the method comprising mutating a basic region/leucine zipper (bZIP)
type transcription factor binding site on a promoter of a nicotine
N-demethylase (NND).
10. The method of claim 9, wherein the NND is selected from the
group consisting of CYP82E4v1, CYP82E5v2, and CYP82E10.
11. The method of claim 10, wherein the NND is CYP82E4v1.
12. The method of claim 11, wherein the bZIP binding site on the
promoter of CYP82E4v1 is an A/G box with a pre-mutated sequence of
TACGTC.
13. The method of claim 12, wherein the mutated binding site has
the sequence TGCGTC.
14. The method of claim 13, wherein the mutated binding site is
formed by site-directed mutagenesis.
15. A method of decreasing conversion of nicotine to nornicotine,
the method comprising mutating a plant genome to knockout at least
one basic region/leucine zipper (bZIP) type transcription
factor.
16. The method of claim 15, wherein the at least one bZIP
transcription factor is selected from the group consisting of group
C bZIP transcription factor, group S bZIP transcription factor, and
combinations thereof.
17. The method of claim 15, wherein the at least one bZIP
transcription factor is selected from the group consisting of
NtbZIP1a, NtbZIP1b, NtbZIP2a, NtbZIP2b, and combinations
thereof.
18. The method of claim 15, wherein the at least one bZIP
transcription factor is selected from the group consisting of
NtbZIP1a and NtbZIP2a, NtbZIP1b and NtbZIP2b, and combinations
thereof.
19. The method of claim 15, further comprising administering at
least one bZIP type transcription factor inhibitor to an organism
in need thereof.
20. The method of claim 19, wherein the at least one bZIP
transcription factor inhibitor is selected from the group
consisting of an NtbZIP1a inhibitor, an NtbZIP1b inhibitor, an
NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations
thereof.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/595,983, filed Dec. 7, 2017, the entire
disclosure of which is incorporated herein by this reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. The ASCII copy of the
Sequence Listing, which was created on Dec. 4, 2018, is named
13177N-2183US.txt and is 12 kilobytes in size.
TECHNICAL FIELD
[0003] The present invention relates to articles and methods for
regulating conversion of nicotine to nomicotine. In particular, the
presently-disclosed subject matter relates to transcription factors
for regulating conversion of nicotine to nornicotine and methods of
use thereof.
BACKGROUND
[0004] Nicotiana tabacum (common tobacco) is a natural
allotetraploid originated about 200,000 years ago. The maternal
S-genome is derived from ancestors of N. sylvestris and paternal
T-genome from the relatives of N. tomentosiformis. Nicotine is the
major alkaloid accumulated in most of the cultivated tobacco
varieties. During the past decades, significant progress has been
made in isolation and characterization structural genes in the
nicotine biosynthetic pathway. Jasmonic acid (JA) is a major
elicitor of nicotine biosynthesis. JAresponsive transcription
factors (TFs) belong to two major families, APETALA2/ETHYLENE
RESPONSE FACTORS (AP2/ERFs) and the basic HELIX-LOOP-HELIX (bHLH),
and are known to induce the expression of genes encoding key
enzymes in the nicotine biosynthetic pathway.
[0005] Nicotine and other tropane alkaloids, such as hyoscyamine
and scopolamine, are synthesized in roots, and transported through
xylem to the leaves. A number transporters have been isolated and
characterized for their role in transport and vacuolar
sequestration of alkaloids in plants. In tobacco, a number of
transporters belonging to the MULTIDRUG and TOXIC COMPOUND ETRUSION
(MATE) family, including MATE1/2 and Jasmonate-inducible Alkaloid
Transporter (JAT1/2), are involved in transportation and vacuolar
sequestration of nicotine. However, TFs involved in regulation of
these transporter are not thoroughly studied.
[0006] In addition to nicotine, tobacco plants accumulate three
other pyridine alkaloids namely, nornicotine, anabasine, and
anatabine. Nornicotine is a demethylated nicotine (does not contain
a methyl group) that is derived from nicotine by an enzymatic
process. It is also a precursor to N-nitrosonornicotine (NNN),
which is produced during the curing and processing of tobacco
materials. More specifically, during post-harvest processing,
nornicotine chemically reacts with the nitrosating agents to form
NNN. As NNN belongs to a class of smoking related carcinogens
called tobacco specific nitrosamines (TSNA), it is highly desirable
to reduce TSNA in tobacco products.
[0007] There are two possible ways to reduce TSNA. One is to reduce
overall nicotine content; the other is to eliminate conversion of
nicotine to nornicotine. Conversion of nicotine to nornicotine is
catalyzed by nicotine N-demethylase (NND), a small family of
cytochrome P450 enzymes. Three NND genes, CYP82E4v1 (originated
from N. tomentosiformis), CYP82E5v2 (originated from N.
tomentosiformis), and CYP82E10 (originated from N. sylvestris),
have been identified in the conversion of nicotine to nornicotine
in tobacco. CYP82E4v1 (E4) plays a major role in nicotine to
nornicotine conversion in senescent leaves, while expression of
CYP82E10 (E10) is reported to be in the roots and CYP82E5 (E5)
functions in both roots and leaves. However, up to this point,
transcription factors (TFs) involved in the regulation of nicotine
to nornicotine conversion (i.e., transcriptional regulators of E4,
5, and 10 genes) have not been identified. Therefore, although
significant progress has been made in biochemical and molecular
characterization of these nicotine transporters and enzymes
involved in nornicotine biosynthesis, the molecular mechanism
underlying the regulation of these genes remains to be
elucidated.
[0008] Accordingly, there is a need for articles and methods that
regulate the conversion of nicotine to nornicotine.
SUMMARY
[0009] The presently-disclosed subject matter meets some or all of
the above-identified needs, as will become evident to those of
ordinary skill in the art after a study of information provided in
this document.
[0010] This summary describes several embodiments of the
presently-disclosed subject matter, and in many cases lists
variations and permutations of these embodiments. This summary is
merely exemplary of the numerous and varied embodiments. Mention of
one or more representative features of a given embodiment is
likewise exemplary. Such an embodiment can typically exist with or
without the feature(s) mentioned; likewise, those features can be
applied to other embodiments of the presently-disclosed subject
matter, whether listed in this summary or not. To avoid excessive
repetition, this summary does not list or suggest all possible
combinations of such features.
[0011] In some embodiments, the presently-disclosed subject matter
includes a method of decreasing conversion of nicotine to
nomicotine, the method comprising administering at least one basic
region/leucine zipper (bZIP) type transcription factor inhibitor to
an organism in need thereof. In some embodiments, the at least one
bZIP transcription factor inhibitor is selected from the group
consisting of group C bZIP transcription factor inhibitors, group S
bZIP transcription factor inhibitors, and combinations thereof. In
some embodiments, the at least one bZIP transcription factor
inhibitor is selected from the group consisting of an NtbZIP1a
inhibitor, an NtbZIP1b inhibitor, an NtbZIP2a inhibitor, an
NtbZIP2b inhibitor, and combinations thereof. In some embodiments,
the at least one bZIP transcription factor inhibitor comprises an
NtbZIP1a inhibitor and an NtbZIP1b inhibitor. In some embodiments,
the at least one bZIP transcription factor inhibitor comprises an
NtbZIP2a inhibitor and an NtbZIP2b inhibitor. In some embodiments,
the at least one bZIP transcription factor inhibitor comprises an
NtbZIP1a inhibitor, an NtbZIP1b inhibitor, an NtbZIP2a inhibitor,
and an NtbZIP2b inhibitor.
[0012] In some embodiments, the at least one bZIP transcription
factor inhibitor is selected from the group consisting of antisense
oligonucleotides, miRNA, siRNA, locked nucleic acid (LNA)
nucleotides, and combination thereof. In one embodiment, the at
least one bZIP transcription factor inhibitor comprises an
antisense oligonucleotide of a bZIP transcription factor selected
from the group consisting of NtbZIP1a, NtbZIP1b, NtbZIP2a,
NtbZIP2b, and combinations thereof.
[0013] Also provided herein, in some embodiments, is a method of
decreasing conversion of nicotine to nornicotine, the method
comprising mutating a basic region/leucine zipper (bZIP) type
transcription factor binding site on a promoter of a nicotine
N-demethylase (NND). In some embodiments, the NND is selected from
the group consisting of CYP82E4v1, CYP82E5v2, and CYP82E10. In one
embodiment, the NND is CYP82E4v1. In another embodiment, the bZIP
binding site on the promoter of CYP82E4v1 is an A/G box with a
pre-mutated sequence of TACGTC. In a further embodiment, the
mutated binding site has the sequence TGCGTC. In some embodiments,
the mutated binding site is formed by site-directed mutagenesis. In
some embodiments, the method also includes administering at least
one bZIP type transcription factor inhibitor to an organism in need
thereof. In one embodiment, the at least one bZIP transcription
factor inhibitor is selected from the group consisting of an
NtbZIP1a inhibitor, an NtbZIP1b inhibitor, an NtbZIP2a inhibitor,
an NtbZIP2b inhibitor, and combinations thereof.
[0014] Further provided herein, in some embodiments, is a method of
decreasing conversion of nicotine to nornicotine, the method
comprising mutating a plant genome to knockout at least one basic
region/leucine zipper (bZIP) type transcription factor. In some
embodiments, the at least one bZIP transcription factor is selected
from the group consisting of group C bZIP transcription factor,
group S bZIP transcription factor, and combinations thereof. In
some embodiments, the at least one bZIP transcription factor is
selected from the group consisting of NtbZIP1a, NtbZIP1b, NtbZIP2a,
NtbZIP2b, and combinations thereof. In some embodiments, the at
least one bZIP transcription factor is selected from the group
consisting of NtbZIP1a and NtbZIP2a, NtbZIP1b and NtbZIP2b, and
combinations thereof. In some embodiments, the method also includes
administering at least one bZIP type transcription factor inhibitor
to an organism in need thereof. In one embodiment, the at least one
bZIP transcription factor inhibitor is selected from the group
consisting of an NtbZIP1a inhibitor, an NtbZIP1b inhibitor, an
NtbZIP2a inhibitor, an NtbZIP2b inhibitor, and combinations
thereof.
[0015] Further features and advantages of the presently-disclosed
subject matter will become evident to those of ordinary skill in
the art after a study of the description, figures, and non-limiting
examples in this document.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The presently-disclosed subject matter will be better
understood, and features, aspects and advantages other than those
set forth above will become apparent when consideration is given to
the following detailed description thereof. Such detailed
description makes reference to the following drawings, wherein:
[0017] FIG. 1 shows a graph illustrating hierarchical cluster
analysis of the transcriptome data of eight different tobacco
tissues. Each tissue forms a distinct cluster based on the
expression.
[0018] FIG. 2 shows a graph illustrating distribution of different
TF families in tobacco and its progenitors.
[0019] FIG. 3 shows a graph illustrating co-expression analysis of
TF genes, and structural genes in nicotine biosynthetic pathway in
different tissue. The TF and structural genes are grouped into 8
different modules (color-coded side bar) based on their expression.
The black module contains majority of the structural gene nicotine
biosynthetic pathway. YF, young flower; MF, mature flower; YL,
young leaf; ML, mature leaf SL, senesce leaf.
[0020] FIG. 4 shows a graph illustrating expression of NtbZIP1a/b
and NNDs in different tobacco tissues.
[0021] FIG. 5 shows an image illustrating the bZIP family in
tobacco. NtbZIP1a and NtbZIP1b are indicated by *.
[0022] FIG. 6 shows an image comparing the nucleotide and amino
acid sequences of NtbZIP1a and 1b.
[0023] FIG. 7 shows a graph illustrating that transient
overexpression of NtbZIP1a in tobacco leaves induces the expression
of CYP82E4v1, CYP82E5v2, and CYP82E10.
[0024] FIG. 8 shows a graph illustrating that NtbZIP1a
significantly activates CYP82E4v1 and CYP82E10 promoters in tobacco
cells.
[0025] FIG. 9 shows a graph illustrating that NtbZIP1a activates
CYP82E4v1 promoter in tobacco cells by binding to the A/G box.
[0026] FIG. 10 shows a graph illustrating that NtbZIP1a and b
significantly activate CYP82E4v1 promoter in tobacco cells.
[0027] FIG. 11 shows a graph illustrating that topping of tobacco
plants downregulates the expression of NtbZIPs and NNDs.
[0028] FIGS. 12A-C show graphs and images illustrating
overexpression of NtbZIP1a in tobacco plants. (A) Genomic DNA PCR
and cDNA PCR of control and three transgenic lines (line #4, 5, and
9) confirming the integration and expression, respectively, of the
antibiotic selection marker, neomycin phosphotransferase II (npt
II; Kan). (B) Quantitative real-time (qRT-PCR) analysis showing the
relative expression of NtbZIP1 and E4 in control (EV) and
transgenic lines (line #4, 5, and 9). (C) Metabolic analysis
showing conversion of nicotine to nornicotine in control and
transgenic lines (To or first generation transgenic plants).
[0029] FIG. 13 shows an image illustrating amino acid sequence
alignment of NtbZIP2a and 2b.
[0030] FIG. 14 shows a graph illustrating that NtbZIP2a and b have
similar expression patterns in tobacco tissues.
[0031] FIG. 15 shows a graph illustrating that NtbZIP2 acts
synergistically with NtbZIP1 to activate the E4 promoter in tobacco
cells.
[0032] FIGS. 16A-B show images illustrating protein-protein
interaction of NtbZIP1 and NtbZIP2 using yeast two hybrid assay.
(A) Yeast two hybrid assay showing protein-protein interaction
between NtbZIP1 and NtbZIP2. Colony growth on synthetic drop-out
(SD) medium lacking leucine, tryptophan, histidine and adenine
(-leu-trp-his-ade) indicates interaction between the proteins
(bZIPs). (B) Schematic diagram of NtbZIP1 and NtbZIP2. The bZIP
domain is indicated by "shaded" rectangle. The numbers indicate the
amino acid.
[0033] While the disclosure is susceptible to various modifications
and alternative forms, specific embodiments thereof have been shown
by way of example in the drawings and are herein described below in
detail. It should be understood, however, that the description of
specific embodiments is not intended to limit the disclosure to
cover all modifications, equivalents and alternatives falling
within the spirit and scope of the disclosure as defined by the
appended claims.
Definitions
[0034] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure belongs. Any
methods and materials similar to or equivalent to those described
herein can be used in the practice or testing of the present
disclosure, including the methods and materials are described
below.
[0035] Following long-standing patent law convention, the terms
"a," "an," and "the" refer to "one or more" when used in this
application, including the claims. Thus, for example, reference to
"a cell" includes a plurality of cells, and so forth.
[0036] The terms "comprising," "including," and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
[0037] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as reaction conditions,
and so forth used in the specification and claims are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated to the contrary, the numerical
parameters set forth in this specification and claims are
approximations that can vary depending upon the desired properties
sought to be obtained by the presently-disclosed subject
matter.
[0038] As used herein, the term "about," when referring to a value
or to an amount of mass, weight, time, volume, concentration,
percentage, or the like is meant to encompass variations of in some
embodiments .+-.50%, in some embodiments .+-.40%, in some
embodiments .+-.30%, in some embodiments .+-.20%, in some
embodiments .+-.10%, in some embodiments .+-.5%, in some
embodiments .+-.1%, in some embodiments .+-.0.5%, and in some
embodiments .+-.0.1% from the specified amount, as such variations
are appropriate to perform the disclosed method.
[0039] As used herein, ranges can be expressed as from "about" one
particular value, and/or to "about" another particular value. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0040] All combinations of method or process steps as used herein
can be performed in any order, unless otherwise specified or
clearly implied to the contrary by the context in which the
referenced combination is made.
DETAILED DESCRIPTION
[0041] The details of one or more embodiments of the
presently-disclosed subject matter are set forth in this document.
Modifications to embodiments described in this document, and other
embodiments, will be evident to those of ordinary skill in the art
after a study of the information provided in this document. The
information provided in this document, and particularly the
specific details of the described exemplary embodiments, is
provided primarily for clearness of understanding and no
unnecessary limitations are to be understood therefrom. In case of
conflict, the specification of this document, including
definitions, will control.
[0042] The presently-disclosed subject matter relates to articles
and methods for regulating conversion of nicotine to nornicotine.
In some embodiments, the article includes one or more transcription
factor (TF) inhibitors. In one embodiment, for example, the article
includes one or more inhibitors of basic region/leucine zipper
(bZIP) type transcription factors. In another embodiment, the bZIP
type transcription factors are derived from tobacco. In a further
embodiment, of the 133 bZIP type transcription factors identified
in tobacco by the instant inventors, which are classified into ten
sub-groups: A, B, C, D, E, F, G, H, and S, suitable bZIP type
transcription factors include at least one of the 27 bZIPs in
sub-group S, at least one of the 6 bZIPs in sub-group C, other bZIP
63 homologs, or a combination thereof. In certain embodiments, the
S sub-group bZIP transcription factor includes, but is not limited
to, NtbZIP1a (SEQ ID NOs: 1 and 2), NtbZIP1b (SEQ ID NOs: 3 and 4),
or a combination thereof; and/or the C sub-group bZIP transcription
factor includes, but is not limited to, NtbZIP2a (SEQ ID NO: 5),
NtbZIP2b (SEQ ID NO: 6), or a combination thereof.
[0043] The one or more transcription factor inhibitors include, but
are not limited to, antisense oligonucleotides, miRNA, siRNA,
locked nucleic acid (LNA) nucleotides, or a combination thereof. In
some embodiments, the inhibitors provide RNAi-mediated
knock-down/silencing of the bZIP type transcription factors. As
will be appreciated by those skilled in the art, the specific
sequence/structure of the transcription factor inhibitors is based
upon the sequence of the specific transcription factor.
Accordingly, as will also be appreciated by those skilled in the
art, the antisense oligonucleotides and/or LNAs may be formed by
any suitable method using the bZIP type transcription factor
sequences provided herein. For example, in one embodiment, the
inhibitor includes an antisense oligonucleotide having 100%
sequence homology with the complementary bZIP type transcription
factor. In another embodiment, the transcription factor inhibitor
includes an antisense oligonucleotide having 100% sequence homology
with a bZIP type transcription factor complementary to NtbZIP1a,
NtbZIP1b, NtbZIP2a, and/or NtbZIP2b. In such embodiments, the
transcription factor inhibitor(s) provide RNAi-mediated
knock-down/silencing of NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or
NtbZIP2b expression in tobacco.
[0044] Also provided herein, in some embodiments, is a method of
regulating the conversion of nicotine to nornicotine in a tobacco
plant or other nicotine containing organism. In one embodiment, the
method includes administering one or more of the bZIP inhibitors
disclosed herein to a nicotine containing organism. Administration
of these one or more bZIP inhibitors decreases or eliminates
conversion of the nicotine to nornicotine. The one or more
inhibitors may be administered for a single type of bZIP
transcription factor, or for a combination of bZIP transcription
factors. For example, in one embodiment, the method includes
administering one or more inhibitors for S bZIP type transcription
factors or C bZIP type transcription factors. In another
embodiment, the method includes administering one or more
inhibitors for S bZIP type transcription factors and one or more
transcription factors for C bZIP type transcription factors. In a
further embodiment, the method includes administering one or more
inhibitors of NtbZIP1a, NtbZIP1b, NtbZIP2a, and/or NtbZIP2b to the
organism. In certain embodiments, inhibiting both S bZIP type
transcription factors and C bZIP type transcription factors has a
synergistic effect on the reduction or elimination of nicotine
conversion to nornicotine.
[0045] The methods disclosed herein include administering a single
type of inhibitor or any suitable combination of inhibitors, which
may be the same or different for each bZIP transcription factor
being inhibited. For example, in one embodiment, the method
includes administering antisense oligonucleotides of NtbZIP1a,
NtbZIP1b, NtbZIP2a, and/or NtbZIP2b. In another embodiment, the
method includes administering antisense oligonucleotides of one
bZIP transcription factor, such as NtbZIP1a, and LNA nucleotides of
another bZIP transcription factor, such as NtbZIP1b. As will be
appreciated by those skilled in the art, although discussed above
with regard to certain combinations of bZIP transcription factors
and transcription factor inhibitors, the disclosure is not so
limited and may include any other suitable combination of TFs and
TF inhibitors.
[0046] Additionally or alternatively, the method may include bZIP
type transcription factor knockout and/or mutation of a bZIP type
transcription factor binding site on the promoter of the nicotine
N-demethylase (NND). For example, in one embodiment, the method
includes editing the plant genome to knock-out NtbZIP1a, NtbZIP1b,
NtbZIP2a, and/or NtbZIP2b. The genome editing may be performed
through any suitable process, such as, but not limited to,
CRISPR/Cas9-mediated genome editing. In another embodiment, the
method includes mutating the bZIP binding element in the E4
promoter, called A/G box (TACGTC), to TGCGTC by site-directed
mutagenesis. Although discussed above with regard to a specific
mutation in the E4 promoter, as will be appreciated by those
skilled in the art, the disclosure is not so limited and includes
any other mutation in the E4, E5, and/or E10 promoter to reduce or
eliminate activation of the respective NND by the bZIP type
transcription factor.
[0047] The administration of the TF inhibitors, the TF knockout,
and/or the binding site mutation disclosed herein reduces or
eliminates activation of the NND by the bZIP type transcription
factor, which decreases or eliminates conversion of nicotine to
nornicotine. As opposed to existing articles that include E4, E5,
and E10 mutants, the articles disclosed herein control the
expression of E4, E5, and E10 to reduce or eliminate the conversion
of nicotine to nornicotine. By reducing or eliminating the
conversion of nicotine to nornicotine the articles and methods
disclosed herein decrease the harmful effects of products which
typically contain the carcinogenic nornicotine, such as, but not
limited to, tobacco products.
[0048] The presently-disclosed subject matter is further
illustrated by the following specific but non-limiting examples.
The following examples may include compilations of data that are
representative of data gathered at various times during the course
of development and experimentation related to the
presently-disclosed subject matter.
EXAMPLES
Example 1
[0049] This Example describes the analysis of transcriptome data
sets of different tobacco tissues, including leaf (young, mature,
and senesce leaf), root, stem, flower (young and mature flower),
and capsule to generate a co-expression network. First, a
hierarchical cluster analysis was performed, which revealed that
each individual tissue type exhibits a unique expression pattern
(FIG. 1). Next, the genes encoding all major TF families in tobacco
and its progenitors were identified. The tobacco genome contains
more TF genes than its progenitors and the number of TFs belonging
to MYB, AP2/ERFs, and bHLH families are significantly higher than
other families (FIG. 2). In view thereof, the TFs and structural
genes in the nicotine biosynthetic pathway were then grouped into 8
different modules (color-coded side bar) based on their expression
pattern in different tissues (FIG. 3). The "black" module is
particularly interesting as majority of the nicotine biosynthetic
genes were found in this module along with a number of TF genes.
Many of these TFs belong to MYB, bHLH, bZIP, and ERF families. As
discussed in Examples 2-4 below, the role of these TFs in nicotine
biosynthesis in tobacco is established through isolation and
functional characterization thereof.
Example 2
[0050] This Example describes two bZIP type transcription factors
from tobacco (FIG. 4), which regulate the conversion of nicotine to
nornicotine, can be used for reduction of smoking related
carcinogen, tobacco specific nitrosamines (TSNA).
[0051] bZIP TFs are characterized by a conserved leucine zipper
motif that mediates dimer formation for DNA binding. In plants,
bZIP TFs regulate processes including pathogen defense, light and
stress signaling, seed maturation, and flower development. Many
bZIP factors, especially those in tobacco, are not well
characterized. By co-expression and clustering analyses, two bZIP
TF genes that co-express with E4, E5, and E10 were identified
herein. These tobacco bZIP TFs have been termed NtbZIP1a and
NtbZIP1b.
[0052] NtbZIP1a/b exhibit similar expression patterns as compared
to CYP82E4v1, the major NND enzyme involved in nicotine to
nornicotine conversion, and are highly expressed in flowers and
senescent leaves (FIG. 4). As illustrated in FIG. 5, 133 bZIPs were
identified in tobacco and classified into ten sub-groups: A, B, C,
D, E, F, G, H, I, and S, with NtbZIP1 a/b belonging to sub-group S.
In maize, expression of group-S bZIPs are induced by wounding,
cold, and drought stress. Referring to FIG. 6, it was also found
that NtbZIP1a and b are more than 97% identical at nucleotide and
amino acid level. Without wishing to be bound by theory, it is
believed that the two homologous bZIPs are derived from two
progenitors of tobacco, N. sylvestris and N. tometosiformis.
[0053] After identifying the two bZIP TFs, whether overexpression
of NtbZIP1a leads to upregulation of E4, 5, and 10 was tested.
NtbZIP1a was cloned into pCAMBIA2300 (binary vector) under the
control of the CaMV 35S promoter and rbcS terminator. The binary
vectors (empty control and NtbZIP1a) were mobilized into
Agrobacterium, and tobacco leaves were infiltrated using the
transformed Agrobacterium. Total RNA isolated from
Agrobacterium-infiltrated leaf discs were used for cDNA synthesis
and real-time quantitative PCR (qRT-PCR) was used to detect the
transcript levels of NtbZIP1a, NtbZIP1b, E4, 5, and 10. An
ubiquitously expressed house-keeping gene, tubulin, was used as
internal control in qRT-PCR. The results showed that, when NtbZIP1a
was highly expressed transiently, the endogenous NtbZIP1b, E4, 5,
and 10 were upregulated (approx. 3-10 fold), indicating that
NtbZIP1a induces the expression of NtbZIP1b, E4, 5, and 10, hence a
possible transcriptional activator for these genes (FIG. 7).
[0054] Next, whether NtbZIP1a can bind to the promoters of its
potential target genes was tested. The promoters (approximately 1.0
kb fragment of the 5' untranslated region of each coding gene) of
E4, 5, and 10 were isolated, and the individual promoters were
fused to a firefly luciferase reporter gene. The NtbZIP1a gene was
cloned into the pBlueScript (pBS) vector under the control of the
CaMV 35S promoter and rbcS terminator. The plasmids were
electroporated into tobacco protoplasts. NtbZIP1a significantly
induced the luciferase gene expression controlled by the E4 and E10
promoters, but not E5 promoter, suggesting that NtbZIP1a can
directly activate E4 and E10 genes, likely by binding to their
promoters (FIG. 8). NtbZIP1a did not appear to bind to the 1.0 kb
promoter region of E5, used for the activation experiment. However,
as mentioned above, overexpression of NtbZIP1a led to upregulation
of E5, together with E4 and 10. The promoter activation experiment
indicates two possible NtbZIP1a regulatory relationships with E5
gene: (1) NtbZIP1a binds to a site outside of the 1.0 kb promoter
fragment, or (2) NtbZIP1a indirectly activates E5, through another
unidentified activator in tobacco (e.g., NtbZIP1a activates another
activator, which in turn activates E5).
[0055] To support the possibility of NtbZIP1a directly binding to
the E4 promoter to regulate transactivation the bZIP binding
element in the E4 promoter, called A/G box (TACGTC), was mutated to
TGCGTC by site-directed mutagenesis. The mutated promoter was fused
to the luciferase reporter gene as described above. A
transactivation experiment was then performed using the mutant
reporter plasmid and the NtbZIP1a expression vector, as described
above. The result showed that NtbZIP1a is unable to activate the
luciferase gene expression under the control of the mutant promoter
(FIG. 9). This experiment demonstrated that the E4 promoter is
activated through an A/G-box binding factor, most likely
NtbZIP1a.
[0056] Referring to FIG. 10, the transactivation experiment was
also performed using a NtbZIP1b expression vector and the E4
promoter-luciferase reporter plasmid, as described for NtbZIP1a.
NtbZIP1b also activated the E4 promoter at the similar level as
NtbZIP1a.
[0057] Finally, as it has been established that the agronomic
practice of tobacco topping (removal of the axillary shoots)
induces nicotine production, the instant inventors analyzed the
transcriptome data from tobacco plants that were topped or
un-topped. More specifically, leaf samples were collected after 24
hours from the control (un-topped) and topped plants. RNA isolated
from un-topped and topped leaves were used for cDNA synthesis and
qRT-PCR analyses. The results showed that topping resulted in
decreased expression of bZIP1a/b, as well as E4, 5, and 10, by
approximately 70-90%, compared to the un-topped plants, suggesting
that topping negatively regulates the expression of bZIP1a/b and
NNDs in tobacco (FIG. 11). The result also indicates that bZIP1a/b
and NNDs are coordinately expressed in tobacco, as gene regulators
and their target genes usually do.
[0058] Based upon the Example above, NtbZIP1a and 1b are believed
to be involved in the regulation of the three NND genes as
activators. Reduction or inactivation of NtbZIP1a/b may lead to
reduction of nornicotine.
Example 3
[0059] This Example describes the formation of transgenic lines
overexpressing NtbZIP1a and the effect of these transgenic lines on
endogenous E4 expression.
[0060] To form the transgenic lines, the pCAMBIA2300 (binary
vector), containing NtbZIP1a under the control of the CaMV 35S
promoter and rbcS terminator, was mobilized into Agrobacterium, and
tobacco leaf discs were infected with the transformed
Agrobacterium. More than 20 transgenic lines overexpressing
NtbZIP1a were generated from Agrobacterium-infected leaf discs.
Genomic DNA isolated from control and three transgenic lines were
used to verify the transgenic status of the plants by PCR
amplification of the antibiotic selection marker, neomycin
phophotransferase II (nptII; kan). Total RNA isolated from leaves
of control and three transgenic lines were used for cDNA synthesis.
RT-PCR was used to verify the expression of nptII (kan) gene in the
transgenic plants (FIG. 12A). Real-time quantitative PCR (qRT-PCR)
was used to detect the transcript levels of NtbZIP1a, and E4 (FIG.
12B). An ubiquitously expressed house-keeping gene, tubulin, was
used as internal control in qRT-PCR.
[0061] The results of this Example showed that NtbZIP1a expression
was significantly higher in the transgenic plants compared with
control. When NtbZIP1a was highly expressed the endogenous E4
expression was unregulated (approx. 6-8 fold), indicating that
NtbZIP1a induces the expression of E4 and therefore is a possible
transcriptional activator for E4 gene. Additionally, metabolic
analysis shows that nicotine to nornicotine conversion is higher in
transgenic tobacco leaves as compared with a control (FIG. 12C).
The formula used for calculating the conversion of nicotine to
nornicotine was:
Nicotine conversion = Nornicotine Nicotine + Nornicotine .times.
100 ##EQU00001##
[0062] Two of the three lines analyzes showed higher nicotine to
nornicotine conversion. Because the metabolic analysis was
performed with independent T.sub.0 (first generation transgenic
plants) segregating population, the metabolic outcomes can
vary.
Example 4
[0063] As described in Example 2 above, the instant inventors have
characterized NtbZIP1a and b, two NtbZIP belonging to group S bZIP
factors. In Arabidopsis, group S bZIP factors are known to interact
with certain group C factors to regulate gene expression. In view
of this interaction, the instant inventors identified the tobacco
homologs of Arabidopsis bZIP 63, a group C member that interacts
with group S factors. In tobacco, there are two bZIP 63 homologs,
termed here as NtbZIP2a and b, that share greater than 95% in amino
acid identity (FIG. 13). Without wishing to be bound by theory, it
is believed that NtbZIP2a and b are originated from the two tobacco
progenitors, N. sylvestris and N. tometosiformis, and functionally
redundant.
[0064] According to transcriptomic analysis, NtbZIP2a and b have
similar expression patterns in tobacco flowers, leaves, stems, and
roots (FIG. 14). Based upon the foregoing discussion, the instant
inventors hypothesized that NtbZIP2a and b also regulate E4/5/10,
individually and/or cooperatively with NtbZIP1a and b. Thus,
NtbZIP2a was tested for transactivation of the E4 promoter,
individually or in combination with NtbZIP1a. More specifically,
the E4 promoter was fused to the firefly luciferase reporter gene
and the bZIP TFs were cloned into pBS vector under the control of
the CaMV35S promoter and rbcs terminator. The results showed that,
individually, both NtbZIP1a and NtbZIP2a activate the E4 promoter;
however, when both NtbZIP1a and 2a were co-expressed,
transactivation of the E4 promoter was significantly increased,
compared to that was induced by each factor alone (FIG. 15).
[0065] Next, to determine whether NtbZIP1a/b interact with
NtbZIP2a, the inventors performed yeast hybrid assay. The growth of
the yeast cells on synthetic drop-out (SD) medium lacking leucine,
trptophan, histidine, and adenine (SD-leu-trp-his-ade) suggests
that NtbZIP1a/b interact with NtbZIP2a (FIG. 16A; 1 and 2). In
addition, NtbZIP2 interacts with itself to form a homo-dimer (FIG.
16A; 3). The bZIP domains of NtbZIP1 and NtbZIP2 are illustrated in
FIG. 16B.
[0066] Although the instant Example only tested NtbZIP2a activity
on the E4 promoter, but not the E5 or E10 promoters, it is believed
that both NtbZIP2 factors are activators of E4/5/10 genes. That is,
this Example suggests that the group C NtbZIP2a and b are two
previously uncharacterized regulators of E4/5/10 genes. In
addition, this Example shows that NtbZIP1 and NtbZIP2 are
synergistic in activation of the E4 (possibly E5 and 10) promoter.
In particular, without wishing to be bound by theory, it is
believed that NtbZIP2a and/or b interact with NtbZIP1a and/or b to
enhance DNA binding ability and significantly increase activation
of the E4/5/10 promoters, as compared to NtbZIP1 or NtbZIP2
alone.
[0067] In summary, two group S and two group C NtbZIP factors that
are positive regulators of E4/5/10 genes were characterized herein.
The transactivation activities of NtbZIP1a and 2a on the E4
promoter are additive. Therefore, without wishing to be bound by
theory, it is believed that knockout approaches to inactivate one
or all of these genes reduces E4/5/10 gene expression.
REFERENCES
[0068] [1] Gavilano, L. B., and Siminszky, B. (2007). Isolation and
characterization of the cytochrome P450 gene CYP82E5v2 that
mediates nicotine to nornicotine conversion in the green leaves of
tobacco. Plant & cell physiology 48, 1567-1574. [0069] [2]
Higo, K., Ugawa, Y., Iwamoto, M., and Higo, H. (1998). PLACE: a
database of plant cis-acting regulatory DNA elements. Nucleic acids
research 26, 358-359. [0070] [3] Leitch, I. J., Hanson, L., Lim, K.
Y., Kovarik, A., Chase, M. W., Clarkson, J. J., and Leitch, A. R.
(2008). The ups and downs of genome size evolution in polyploid
species of Nicotiana (Solanaceae). Annals of botany 101, 805-814.
[0071] [4] Lescot, M., Dehais, P., Thijs, G., Marchal, K., Moreau,
Y., Van de Peer, Y., Rouze, P., and Rombauts, S. (2002). PlantCARE,
a database of plant cis-acting regulatory elements and a portal to
tools for in silico analysis of promoter sequences. Nucleic acids
research 30, 325-327. [0072] [5] Lewis, R. S., Bowen, S. W., Keogh,
M. R., and Dewey, R. E. (2010). Three nicotine demethylase genes
mediate nornicotine biosynthesis in Nicotiana tabacum L.:
functional characterization of the CYP82E10 gene. Phytochemistry
71, 1988-1998. [0073] [6] Lim, K. Y., Matyasek, R., Kovarik, A.,
and Leitch, A. R. (2004). Genome evolution in allotetraploid
Nicotiana. Biol J. Linn Soc 82, 599-606. [0074] [7] Morita, M.,
Shitan, N., Sawada, K., Van Montagu, M. C., Inze, D., Rischer, H.,
Goossens, A., Oksman-Caldentey, K. M., Moriyama, Y., and Yazaki, K.
(2009). Vacuolar transport of nicotine is mediated by a multidrug
and toxic compound extrusion (MATE) transporter in Nicotiana
tabacum. Proceedings of the National Academy of Sciences of the
United States of America 106, 2447-2452. [0075] [8] Pattanaik, S.,
Werkman, J. R., Kong, Q., and Yuan, L. (2010a). Site-directed
mutagenesis and saturation mutagenesis for the functional study of
transcription factors involved in plant secondary metabolite
biosynthesis. Methods in molecular biology 643, 47-57. [0076] [9]
Pattanaik, S., Kong, Q., Zaitlin, D., Werkman, J. R., Xie, C. H.,
Patra, B., and Yuan, L. (2010b). Isolation and functional
characterization of a floral tissue-specific R2R3 MYB regulator
from tobacco. Planta 231, 1061-1076. [0077] [10] Shoji, T., and
Hashimoto, T. (2011). Tobacco MYC2 regulates jasmonate-inducible
nicotine biosynthesis genes directly and by way of the NIC2-locus
ERF genes. Plant & cell physiology 52, 1117-1130. [0078] [11]
Shoji, T., Kajikawa, M., and Hashimoto, T. (2010). Clustered
transcription factor genes regulate nicotine biosynthesis in
tobacco. The Plant cell 22, 3390-3409. [0079] [12] Shoji, T., Inai,
K., Yazaki, Y., Sato, Y., Takase, H., Shitan, N., Yazaki, K., Goto,
Y., Toyooka, K., Matsuoka, K., and Hashimoto, T. (2009). Multidrug
and toxic compound extrusion-type transporters implicated in
vacuolar sequestration of nicotine in tobacco roots. Plant
physiology 149, 708-718. [0080] [13] Sierro, N., van Oeveren, J.,
van Eijk, M. J., Martin, F., Stormo, K. E., Peitsch, M. C., and
Ivanov, N. V. (2013a). Whole genome profiling physical map and
ancestral annotation of tobacco Hicks Broadleaf. The Plant journal:
for cell and molecular biology 75, 880-889. [0081] [14] Sierro, N.,
Battey, J. N., Ouadi, S., Bovet, L., Goepfert, S., Bakaher, N.,
Peitsch, M. C., and Ivanov, N. V. (2013b). Reference genomes and
transcriptomes of Nicotiana sylvestris and Nicotiana
tomentosiformis. Genome biology 14, R60. [0082] [15] Sierro, N.,
Battey, J. N., Ouadi, S., Bakaher, N., Bovet, L., Willig, A.,
Goepfert, S., Peitsch, M. C., and Ivanov, N. V. (2014). The tobacco
genome sequence and its comparison with those of tomato and potato.
Nature communications 5, 3833. [0083] [16] Siminszky, B., Gavilano,
L., Bowen, S. W., and Dewey, R. E. (2005). Conversion of nicotine
to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a
cytochrome P450 monooxygenase. Proceedings of the National Academy
of Sciences of the United States of America 102, 14919-14924.
[0084] [17] Singh, S. K., Wu, Y., Ghosh, J. S., Pattanaik, S.,
Fisher, C., Wang, Y., Lawson, D., and Yuan, L. (2015).
RNA-sequencing Reveals Global Transcriptomic Changes in Nicotiana
tabacum Responding to Topping and Treatment of Axillary-shoot
Control Chemicals. Scientific reports 5, 18148. [0085] [18] Van
Moerkercke, A., Steensma, P., Schweizer, F., Pollier, J.,
Gariboldi, I., Payne, R., Vanden Bossche, R., Miettinen, K., Espoz,
J., Purnama, P. C., Kellner, F., Seppanen-Laakso, T., O'Connor, S.
E., Rischer, H., Memelink, J., and Goossens, A. (2015). The bHLH
transcription factor BIS1 controls the iridoid branch of the
monoterpenoid indole alkaloid pathway in Catharanthus roseus.
Proceedings of the National Academy of Sciences of the United
States of America 112, 8130-8135. [0086] [19] Yu, F., and De Luca,
V. (2013). ATP-binding cassette transporter controls leaf surface
secretion of anticancer drug components in Catharanthus roseus.
Proceedings of the National Academy of Sciences of the United
States of America 110, 15830-15835. [0087] [20] Ehlert, A., et al.
(2006) Two-hybrid protein--protein interaction analysis in
Arabidopsis protoplasts: establishment of a heterodimerization map
of group C and group S bZIP transcription factors. The Plant
Journal, 46: 890-900.
[0088] While the disclosure is susceptible to various modifications
and alternative forms, specific embodiments thereof have been shown
by way of example in the drawings and are herein described below in
detail. It should be understood, however, that the description of
specific embodiments is not intended to limit the disclosure to
cover all modifications, equivalents and alternatives falling
within the spirit and scope of the disclosure as defined by the
appended claims.
Sequence CWU 1
1
61435DNANicotiana tabacum 1atggctttga cacagcaacc ggctagttca
ggttctgatg gccaacgtta tgccacaaat 60gacgatagaa aacgaaagag aatggagtcc
aaccgtgaat ctgcaaggcg gtcacggatg 120agaaagcagc agcatttgga
ggagttgatg agccaaatga cacagctaca gaatcagaac 180gttctgtggc
gcgagaagat tgatgctgtg ggaagaaact acctcaccct cgatgcggag
240aacaatgtct tgagggctca aatggcagaa ctgactgaac gcttggattc
tctcaattcg 300ctcactcgtt tctgggctga tgctaatgga ctagctgtgg
atatccctga aattcctgac 360actttgcttg agccctggca gcttccttgc
ccaattcaac ccatcactgc ttctgctgat 420atgtttcagt tttga
4352144PRTNicotiana tabacum 2Asn Ala Leu Thr Gln Gln Pro Ala Ser
Ser Gly Ser Asp Gly Gln Arg1 5 10 15Tyr Ala Thr Asn Asp Asp Arg Lys
Arg Lys Arg Met Glu Ser Asn Arg 20 25 30Glu Ser Ala Arg Arg Ser Arg
Met Arg Lys Gln Gln His Leu Glu Glu 35 40 45Leu Met Ser Gln Met Thr
Gln Leu Gln Asn Gln Asn Val Leu Trp Arg 50 55 60Glu Lys Ile Asp Ala
Val Gly Arg Asn Tyr Leu Thr Leu Asp Ala Glu65 70 75 80Asn Asn Val
Leu Arg Ala Gln Met Ala Glu Leu Thr Glu Arg Leu Asp 85 90 95Ser Leu
Asn Ser Leu Thr Arg Phe Trp Ala Asp Ala Asn Gly Leu Ala 100 105
110Val Asp Ile Pro Glu Ile Pro Asp Thr Leu Leu Glu Pro Trp Gln Leu
115 120 125Pro Cys Pro Ile Gln Pro Ile Thr Ala Ser Ala Asp Met Phe
Gln Phe 130 135 1403435DNANicotiana tabacum 3atggcttcga tacagcaacc
agctagttca ggttctgatg gccaacgata tgctatgaac 60gacgatagaa aacgaaagag
aatggagtcc aaccgtgaat ctgcaaggcg gtcacggatg 120aggaagcagc
agcatttgga agagttgatg agccaaatga cacagctaca gaatcagaac
180gttctgtggc gtgagaagat tgatgctgtg ggaagaaact acctgaccct
tgatgcggag 240aacaatgtcc tgagggctca aatggcagaa ctgactgaac
gcttggattc gctcaattcg 300ctcgctcgtt tctgggctga tgctaatgga
ctagctgtgg atatccctga aattccagac 360actttgcttg agccgtggca
gcttccttgc ccaattcaac ccatcactgc ttctgctaat 420atgtttcagt tttga
4354144PRTNicotiana tabacum 4Asn Ala Ser Ile Gln Gln Pro Ala Ser
Ser Gly Ser Asp Gly Gln Arg1 5 10 15Tyr Ala Met Asn Asp Asp Arg Lys
Arg Lys Arg Met Glu Ser Asn Arg 20 25 30Glu Ser Ala Arg Arg Ser Arg
Met Arg Lys Gln Gln His Leu Glu Glu 35 40 45Leu Met Ser Gln Met Thr
Gln Leu Gln Asn Gln Asn Val Leu Trp Arg 50 55 60Glu Lys Ile Asp Ala
Val Gly Arg Asn Tyr Leu Thr Leu Asp Ala Glu65 70 75 80Asn Asn Val
Leu Arg Ala Gln Met Ala Glu Leu Thr Glu Arg Leu Asp 85 90 95Ser Leu
Asn Ser Leu Ala Arg Phe Trp Ala Asp Ala Asn Gly Leu Ala 100 105
110Val Asp Ile Pro Glu Ile Pro Asp Thr Leu Leu Glu Pro Trp Gln Leu
115 120 125Pro Cys Pro Ile Gln Pro Ile Thr Ala Ser Ala Asn Met Phe
Gln Phe 130 135 1405455PRTNicotiana tabacum 5Met Asp Arg Val Phe
Ser Val Asp Asp Asp Ile Gly Asp His Phe Trp1 5 10 15Ser Thr Pro Pro
Thr Ala Asp Leu Gly Val Asp Ser Pro Thr Ala Ala 20 25 30Ala Ala Val
Ser Tyr Ser Lys Met Met Asn Arg Ser Ser Ser Glu Trp 35 40 45Ala Phe
Gln Arg Phe Leu Gln Glu Ala Thr Ala Ala Gly Thr Ser Thr 50 55 60Ser
Ser Pro Pro Gln Pro Pro Thr Met Thr Ala Ser Ser Ser Ser Ser65 70 75
80Ser His Gln Asn Asp Val Val Glu Ile Lys Asp Glu Asn Leu Ser Ile
85 90 95Pro Asn Leu Asn Pro Ser Thr Ala Leu Asn Ser Lys Pro Ala Ser
Ser 100 105 110Phe Gly Leu Ala Pro Pro Pro Asn Ile Ala Val Asp Ser
Glu Glu Tyr 115 120 125Gln Ala Phe Leu Lys Ser Gln Leu His Leu Ala
Cys Ala Ala Val Ala 130 135 140Leu Thr Arg Gly Lys Ser Leu Asn Pro
Gln Asp Ser Gly Ser Thr Ala145 150 155 160His Asp Lys Gly Ser Glu
Thr Ala Ser Ala Ala Gln Ser Gly Ser His 165 170 175Val Ser Thr Leu
Gly Lys Cys Tyr Leu Arg Ser Gly Gln Glu Val Ala 180 185 190Lys Ile
Gln Asp Lys Asp Ala Gly Gly Pro Val Gly Ile Pro Ser Leu 195 200
205Pro Pro Val Gln Lys Lys Pro Val Val Gln Val Arg Ser Thr Thr Ser
210 215 220Gly Ser Ser Arg Glu Gln Ser Asp Asp Asp Glu Ala Glu Gly
Glu Ala225 230 235 240Glu Thr Thr Gln Gly Met Asp Pro Ala Asp Ala
Lys Arg Val Arg Arg 245 250 255Met Leu Ser Asn Arg Glu Ser Ala Arg
Arg Ser Arg Arg Arg Lys Gln 260 265 270Ala His Leu Thr Glu Leu Glu
Thr Gln Val Ser Gln Leu Arg Val Glu 275 280 285Asn Ser Ser Leu Leu
Lys Arg Leu Thr Asp Ile Ser Gln Lys Tyr Asn 290 295 300Glu Ala Ala
Val Asp Asn Arg Val Leu Lys Ala Asp Val Glu Thr Leu305 310 315
320Arg Ala Lys Val Lys Met Ala Glu Glu Thr Val Lys Arg Val Thr Gly
325 330 335Leu Asn Pro Leu Phe Gln Ala Met Ser Glu Ile Ser Ser Met
Val Met 340 345 350Pro Ser Tyr Ser Gly Ser Pro Ser Asp Thr Ser Ala
Asp Ala Ala Val 355 360 365Pro Val Gln Asp Asp Pro Lys His His Tyr
Tyr Gln Gln Pro Pro Asn 370 375 380Asn Leu Met Pro Thr His Asp Pro
Arg Ile Gln Asn Gly Met Val Asp385 390 395 400Val Pro Pro Ile Glu
Asn Val Glu Gln Asn Pro Ala Thr Ala Ala Val 405 410 415Gly Gly Asn
Lys Met Gly Arg Thr Thr Ser Met Gln Arg Val Ala Ser 420 425 430Leu
Glu His Leu Gln Lys Arg Ile Arg Gly Glu Val Ser Ser Cys Gly 435 440
445Thr Gln Gly Arg Gly Glu Gln 450 4556442PRTNicotiana tabacum 6Met
Asp Arg Val Phe Ser Val Asp Asp Asp Ile Gly Asp His Phe Trp1 5 10
15Ser Thr Pro Pro Thr Ala Asp Leu Gly Val Asp Ser Pro Thr Thr Ala
20 25 30Thr Ala Ala Ser Tyr Ser Lys Met Met Asn Arg Ser Ser Ser Glu
Trp 35 40 45Ala Phe Gln Arg Phe Leu Gln Glu Ala Thr Ala Ala Gly Thr
Ser Thr 50 55 60Ser Ser His Pro Gln Pro Pro Thr Met Thr Ala Ser Ser
Ser Ser Ser65 70 75 80Ser His Gln Asn Asp Val Val Glu Ile Lys Asp
Glu Asn Leu Ser Thr 85 90 95Pro Asn Leu Asn Pro Ser Thr Ala Leu Asn
Ser Lys Pro Ala Ser Ser 100 105 110Phe Gly Leu Thr Pro Pro Pro Asn
Ile Ala Val Asp Ser Glu Glu Tyr 115 120 125Gln Ala Phe Leu Lys Ser
Gln Leu His Leu Ala Cys Ala Ala Val Ala 130 135 140Leu Thr Arg Gly
Lys Ser Leu Asn Pro Gln Asp Ser Gly Ser Thr Ala145 150 155 160His
Asp Lys Gly Ser Glu Thr Ala Ser Ala Ala Gln Ser Gly Ser His 165 170
175Glu Met Ala Lys Ile Gln Asp Lys Asp Ala Gly Gly Pro Val Gly Ile
180 185 190Pro Ser Leu Pro Pro Val Gln Lys Lys Pro Val Val Gln Val
Arg Ser 195 200 205Thr Thr Ser Gly Ser Ser Arg Glu Gln Ser Asp Asp
Asp Glu Ala Glu 210 215 220Gly Glu Ala Glu Thr Thr Gln Gly Met Asp
Pro Ala Asp Ala Lys Arg225 230 235 240Val Arg Arg Met Leu Ser Asn
Arg Glu Ser Ala Arg Arg Ser Arg Arg 245 250 255Arg Lys Gln Ala His
Leu Thr Glu Leu Glu Thr Gln Val Ser Gln Leu 260 265 270Arg Val Glu
Asn Ser Ser Leu Leu Lys Arg Leu Thr Asp Ile Ser Gln 275 280 285Lys
Tyr Asn Glu Ala Ala Val Asp Asn Arg Val Leu Lys Ala Asp Val 290 295
300Glu Thr Leu Arg Ala Lys Val Lys Met Ala Glu Glu Thr Val Lys
Arg305 310 315 320Val Thr Gly Leu Asn Pro Leu Phe Gln Ala Met Ser
Glu Ile Ser Ser 325 330 335Met Val Met Pro Ser Tyr Ser Gly Ser Pro
Ser Asp Thr Ser Ala Asp 340 345 350Ala Ala Val Pro Val Gln Asp Asp
Pro Lys His His Tyr Tyr Gln Gln 355 360 365Pro Pro Asn Asn His Met
Pro Thr Asn Asp Pro Arg Ile Gln Asn Gly 370 375 380Met Val Asp Val
Pro Pro Ile Glu Asn Val Gln Gln Asn Pro Ala Thr385 390 395 400Ala
Ala Val Gly Gly Asn Lys Met Gly Arg Thr Ala Ser Met Gln Arg 405 410
415Val Ala Ser Leu Glu His Leu Gln Lys Arg Ile Arg Gly Glu Ile Ser
420 425 430Ser Cys Gly Thr Gln Gly Arg Gly Glu Gln 435 440
* * * * *